Crystallization and preliminary X-ray diffraction analysis of a novel β-L-arabinofuranosidase (HypBA1) fromBifidobacterium longum

Abstract

The -l-arabinofuranosidase (HypBA1) from Bifidobacterium longum JCM 1217 hydrolyzes the -1,2-linked arabinofuranose disaccharide to release l-arabinoses. HypBA1 was classified into glycoside hydrolase family 127 (GH127) by the CAZy website (http://www.cazy.org/). The enzyme was expressed in Escherichia coli and the purified recombinant protein was crystallized. Crystals belonging to the primitive hexagonal space group P3 x 21, with unit-cell parameters a = b = 75.9, c = 254.0 Å , were obtained by the sittingdrop vapour-diffusion method and diffracted to 2.78 Å resolution. A BLASTP search (http://blast.ncbi.nlm.nih.gov/) of the Protein Data Bank did not reveal any similar crystal structures. Structural determination by using SeMet MAD and MIR methods is in progress.

Characterisation of a Hydroxycinnamic Acid Esterase From the Bifidobacterium longum subsp. longum Taxon

“…HCAs are frequently found esterified to plant carbohydrates that are indigestible to the human host and are therefore more likely released in the colon by particular members of the gut microbiota ( Kroon et al, 2000 ; Andreasen et al, 2001 ; Russell et al, 2008 ; McLaughlin et al, 2015 ; Ozcan et al, 2017 ). Much work on plant-derived poly/oligosaccharide metabolism in bifidobacteria has focussed on arabinofuranosidase, xylanase and β-glucosidase activities ( Gueimonde et al, 2007 ; Pokusaeva et al, 2011 ; Fujita et al, 2014 ; Zhu et al, 2014 ; Ku et al, 2015 ). However, since HCAs are a component of plant carbohydrates it is also important to investigate if bifidobacterial produce esterases.…”

β-l-Arabinofurano-cyclitol Aziridines Are Covalent Broad-Spectrum Inhibitors and Activity-Based Probes for Retaining β-l-Arabinofuranosidases

“…However, the presence of a coordination system made of three cysteines and one glutamate centered around a zinc cation within the catalytic domain of HypBA1 12,14 suggested alternative mechanisms to be in play. Site-directed mutagenesis 11 and crystallographic 12,14 cysteine nucleophile, rather than a glutamate or aspartate as in canonical glycosidases. By making use of the mechanism-based inhibitor, β-Larabinofuranosyl cyclophellitol epoxide 15 (1, Figure 1), we recently obtained structural and biochemical evidence for the nature of the nucleophilic residue in HypBA1 and BtGH146, which in both cases turned out to be a cysteine (C417 in HypBA1, C416 in BtGH146) as the nucleophile.…”

“…Crystallographic evidence for the distance (4.9 Å) between two carboxylates (E332 and E338) initially supported a classical two-carboxylate mechanism of action. However, the presence of a coordination system made of three cysteines and one glutamate centered around a zinc cation within the catalytic domain of HypBA1 , suggested alternative mechanisms to be in play. Site-directed mutagenesis and crystallographic , experiments revealed the presence of a cysteine nucleophile, rather than a glutamate or aspartate as in canonical glycosidases.…”

Structure and Catalytic Mechanism of a Glycoside Hydrolase Family-127 β-L-Arabinofuranosidase (HypBA1)

“…The expression and purification methods employed for the protein have been described before [13]. To obtain phase information by multiple isomorphous replacement (MIR), the apo HypBA1 crystals grown in 0.4 M ammonium acetate and 18% w/v polyethylene glycol 3350 [13] were used for preparing heavy atom derived crystals by using the Heavy Atom Screen Hg kit (Hampton Research).…”

“…To obtain phase information by multiple isomorphous replacement (MIR), the apo HypBA1 crystals grown in 0.4 M ammonium acetate and 18% w/v polyethylene glycol 3350 [13] were used for preparing heavy atom derived crystals by using the Heavy Atom Screen Hg kit (Hampton Research). The apo crystals (isomorphous to native crystal) were soaked with various mercurycontaining reagents (final concentration 2 mM) in cryoprotectant solution (0.5 M ammonium acetate, 25% w/v polyethylene glycol 3350 and 5% w/v glycerol) for at least 1 hr.…”